Mesenchymal stem cell culture medium and application thereof

Abstract

The invention discloses a mesenchymal stem cell culture medium and application thereof, and the mesenchymal stem cell culture medium comprises the following raw materials by mass: 50%-70% of a basic culture medium, 20%-30% of a serum-free culture medium, 10%-20% of a special additive, and 1%-5% of an additive. The basic culture medium comprises 32%-40% of serum, 10%-13% of fetal calf serum, 7%-12% of human platelet lysate, 12%-15% of a growth factor supplement, 10%-22% of basic fibroblast growth factors, 10%-18% of epidermal growth factors, 3%-10% of antibiotics, 2%-8% of penicillin, 7%-13% of streptomycin and 6%-10% of L-glutamine; the special additive comprises 10%-21% of an anti-differentiation agent, 10%-22% of an antioxidant and 12%-20% of a directional differentiation inducer; by systematically optimizing culture medium components and culture conditions, the yield and functional consistency of the mesenchymal stem cells can be remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell culture media, and specifically to a mesenchymal stem cell culture medium and its application. Background Art

[0002] Due to its multi-directional differentiation potential, immunomodulatory ability and paracrine effect, mesenchymal stem cells have wide applications in the fields of regenerative medicine, disease treatment and biomanufacturing. The culture medium for mesenchymal stem cells needs to provide a suitable physical and chemical environment, nutritional support and growth signals to maintain their proliferation and multi-directional differentiation potential. Mesenchymal stem cells are a type of adult stem cells with self-renewal and multi-directional differentiation potential, and are widely present in various tissues of fetuses and adults. In addition to self-renewal, mesenchymal stem cells have unique immunomodulatory and tissue regeneration functions. However, the existing mesenchymal stem cell culture media cannot significantly improve the yield and functional consistency of mesenchymal stem cells by systematically optimizing the culture medium components and culture conditions. Therefore, we propose a mesenchymal stem cell culture medium and its application to solve the above-mentioned problems. Summary of the Invention

[0003] The purpose of the present invention is to provide a mesenchymal stem cell culture medium and its application to solve the problems raised in the above background art.

[0004] To achieve the above purpose, the present invention provides the following technical solutions:

[0005] A mesenchymal stem cell culture medium, comprising the following raw materials in parts by mass: 50%-70% of a basal medium, 20%-30% of a serum-free medium, and 10%-20% of a special additive. The basal medium comprises 32%-40% of serum, 10%-13% of fetal bovine serum, 7%-12% of human platelet lysate, 12%-15% of a growth factor supplement, 10%-22% of basic fibroblast growth factor, 10%-18% of epidermal growth factor, 3%-10% of an antibiotic, 2%-8% of penicillin, 7%-13% of streptomycin, and 6%-10% of L-glutamine; the special additive comprises 10%-21% of an anti-differentiation agent, 10%-22% of an antioxidant, and 12%-20% of a directed differentiation inducer.

[0006] As a further solution of the present invention: it further comprises raw materials in the following parts by mass: 40%-70% of a basal medium, 25%-30% of a serum-free medium, and 10%-25% of a special additive. The basal medium comprises 32%-40% of serum, 11%-15% of fetal bovine serum, 6%-15% of human platelet lysate, 10%-16% of a growth factor supplement, 10%-21% of basic fibroblast growth factor, 11%-20% of epidermal growth factor, 4%-8% of an antibiotic, 3%-10% of penicillin, 4.5%-10% of streptomycin, and 5%-10% of L-glutamine; the special additive comprises 10%-20% of an anti-differentiation agent, 10%-20% of an antioxidant, and 10%-20% of a directed differentiation inducer.

[0007] As a further solution of the present invention: it further comprises raw materials in the following parts by mass: 45%-70% of a basal medium, 20%-32% of a serum-free medium, and 12%-20% of a special additive. The basal medium comprises 30%-45% of serum, 10%-16% of fetal bovine serum, 5%-16% of human platelet lysate, 10%-18% of a growth factor supplement, 8%-20% of basic fibroblast growth factor, 12%-20% of epidermal growth factor, 3%-10% of an antibiotic, 2%-8% of penicillin, 5%-10% of streptomycin, and 5%-12% of L-glutamine; the special additive comprises 12%-20% of an anti-differentiation agent, 10%-20% of an antioxidant, and 10%-23% of a directed differentiation inducer.

[0008] As a further solution of the present invention: it further comprises raw materials in the following parts by mass: 50%-65% of a basal medium, 15%-35% of a serum-free medium, and 15%-20% of a special additive. The basal medium comprises 32%-45% of serum, 11%-16% of fetal bovine serum, 5%-15% of human platelet lysate, 9.5%-14.5% of a growth factor supplement, 10%-22% of basic fibroblast growth factor, 12%-20% of epidermal growth factor, 3.5%-8.5% of an antibiotic, 4%-9% of penicillin, 5%-12% of streptomycin, and 3%-10% of L-glutamine; the special additive comprises 13%-20% of an anti-differentiation agent, 15%-20% of an antioxidant, and 12%-20% of a directed differentiation inducer.

[0009] As a further solution of the present invention: It further includes raw materials in the following parts by mass: 35%-60% of basic medium, 25%-40% of serum-free medium, and 20%-30% of special additive. The basic medium includes 30%-40% of serum, 10%-15% of fetal bovine serum, 5%-15% of human platelet lysate, 10%-15% of growth factor supplement, 10%-20% of basic fibroblast growth factor, 10%-20% of epidermal growth factor, 3%-8% of antibiotics, 3%-8% of penicillin, 5%-10% of streptomycin, and 5%-10% of L-glutamine; the special additive includes 10%-20% of anti-differentiation agent, 10%-20% of antioxidant, and 10%-20% of directed differentiation inducer.

[0010] As a further solution of the present invention: The anti-differentiation agent includes ROCK inhibitor.

[0011] As a further solution of the present invention: The antioxidant includes vitamin C.

[0012] As a further solution of the present invention: The directed differentiation inducer includes osteogenic differentiation inducer, adipogenic differentiation inducer, chondrogenic differentiation inducer, and other lineage differentiation inducers.

[0013] As a further solution of the present invention: The osteogenic differentiation inducer includes dexamethasone, β-glycerophosphate, ascorbic acid, and vitamin D3; the adipogenic differentiation inducer includes dexamethasone, 3-isobutyl-1-methylxanthine, insulin, and indomethacin; the chondrogenic differentiation inducer includes TGF-β3, dexamethasone, ascorbic acid, insulin-transferrin-selenium, and sodium pyruvate; the other lineage differentiation inducers include myogenic differentiation inducer and neuron-like differentiation inducer.

[0014] An application of a mesenchymal stem cell medium, where the mesenchymal stem cell medium is used for culturing mesenchymal stem cells.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] For the mesenchymal stem cell medium and its application, the prepared mesenchymal stem cell medium can maintain its proliferation and multi-directional differentiation potential by providing appropriate nutrients and growth factors; maintain its self-renewal ability, multi-directional differentiation potential, and genomic stability; and can significantly improve the yield and functional consistency of mesenchymal stem cells by systematically optimizing the medium components and culture conditions. Specific embodiments

[0017] Example 1

[0018] In one embodiment, a mesenchymal stem cell culture medium comprises raw materials in the following parts by mass: 60% of a basal medium, 25% of a serum-free medium, and 15% of a special additive. The basal medium comprises 36% of serum, 12% of fetal bovine serum, 9% of human platelet lysate, 13% of a growth factor supplement, 18% of basic fibroblast growth factor, 14% of epidermal growth factor, 6% of an antibiotic, 5% of penicillin, 10% of streptomycin, and 8% of L-glutamine; the special additive comprises 18% of an anti-differentiation agent, 18% of an antioxidant, and 15% of a directed differentiation inducer;

[0019] The anti-differentiation agent comprises a ROCK inhibitor; the antioxidant comprises vitamin C; the directed differentiation inducer comprises an osteogenic differentiation inducer, an adipogenic differentiation inducer, a chondrogenic differentiation inducer, and other lineage differentiation inducers; the osteogenic differentiation inducer comprises dexamethasone, β-glycerophosphate, ascorbic acid, and vitamin D3; the adipogenic differentiation inducer comprises dexamethasone, 3-isobutyl-1-methylxanthine, insulin, and indomethacin; the chondrogenic differentiation inducer comprises TGF-β3, dexamethasone, ascorbic acid, insulin-transferrin-selenium, and sodium pyruvate; the other lineage differentiation inducers comprise a myogenic differentiation inducer and a neuron-like differentiation inducer.

[0020] Example 2

[0021] In one embodiment, a mesenchymal stem cell culture medium further comprises raw materials in the following parts by mass: 40% of a basal medium, 25% of a serum-free medium, and 10% of a special additive. The basal medium comprises 34% of serum, 14% of fetal bovine serum, 10% of human platelet lysate, 12% of a growth factor supplement, 16% of basic fibroblast growth factor, 17% of epidermal growth factor, 5% of an antibiotic, 6% of penicillin, 6.5% of streptomycin, and 7.5% of L-glutamine; the special additive comprises 14% of an anti-differentiation agent, 16% of an antioxidant, and 17% of a directed differentiation inducer;

[0022] The anti-differentiation agent comprises a ROCK inhibitor; the antioxidant comprises vitamin C; the directed differentiation inducer comprises an osteogenic differentiation inducer, an adipogenic differentiation inducer, a chondrogenic differentiation inducer, and other lineage differentiation inducers; the osteogenic differentiation inducer comprises dexamethasone, β-glycerophosphate, ascorbic acid, and vitamin D3; the adipogenic differentiation inducer comprises dexamethasone, 3-isobutyl-1-methylxanthine, insulin, and indomethacin; the chondrogenic differentiation inducer comprises TGF-β3, dexamethasone, ascorbic acid, insulin-transferrin-selenium, and sodium pyruvate; the other lineage differentiation inducers comprise a myogenic differentiation inducer and a neuron-like differentiation inducer.

[0023] Example 3

[0024] In one embodiment, a mesenchymal stem cell culture medium further comprises raw materials in the following parts by mass: 70% of a basal medium, 20% of a serum-free medium, and 12% of a special additive. The basal medium comprises 38% of serum, 12% of fetal bovine serum, 10% of human platelet lysate, 13% of a growth factor supplement, 15% of basic fibroblast growth factor, 16% of epidermal growth factor, 5% of an antibiotic, 4% of penicillin, 6% of streptomycin, and 6% of L-glutamine; the special additive comprises 15.5% of an anti-differentiation agent, 16% of an antioxidant, and 18% of a directed differentiation inducer;

[0025] The anti-differentiation agent comprises a ROCK inhibitor; the antioxidant comprises vitamin C; the directed differentiation inducer comprises an osteogenic differentiation inducer, an adipogenic differentiation inducer, a chondrogenic differentiation inducer, and other lineage differentiation inducers; the osteogenic differentiation inducer comprises dexamethasone, β-glycerophosphate, ascorbic acid, and vitamin D3; the adipogenic differentiation inducer comprises dexamethasone, 3-isobutyl-1-methylxanthine, insulin, and indomethacin; the chondrogenic differentiation inducer comprises TGF-β3, dexamethasone, ascorbic acid, insulin-transferrin-selenium, and sodium pyruvate; the other lineage differentiation inducers comprise a myogenic differentiation inducer and a neuron-like differentiation inducer.

[0026] Example 4

[0027] In one embodiment, a mesenchymal stem cell culture medium further comprises raw materials in the following parts by mass: 65% of a basal medium, 15% of a serum-free medium, and 20% of a special additive. The basal medium comprises 40% of serum, 15% of fetal bovine serum, 10% of human platelet lysate, 12.5% of a growth factor supplement, 17% of basic fibroblast growth factor, 14% of epidermal growth factor, 5.5% of an antibiotic, 6.5% of penicillin, 8% of streptomycin, and 6% of L-glutamine; the special additive comprises 15% of an anti-differentiation agent, 17% of an antioxidant, and 15% of a directed differentiation inducer;

[0028] The anti-differentiation agent comprises a ROCK inhibitor; the antioxidant comprises vitamin C; the directed differentiation inducer comprises an osteogenic differentiation inducer, an adipogenic differentiation inducer, a chondrogenic differentiation inducer, and other lineage differentiation inducers; the osteogenic differentiation inducer comprises dexamethasone, β-glycerophosphate, ascorbic acid, and vitamin D3; the adipogenic differentiation inducer comprises dexamethasone, 3-isobutyl-1-methylxanthine, insulin, and indomethacin; the chondrogenic differentiation inducer comprises TGF-β3, dexamethasone, ascorbic acid, insulin-transferrin-selenium, and sodium pyruvate; the other lineage differentiation inducers comprise a myogenic differentiation inducer and a neuron-like differentiation inducer.

[0029] Example 5

[0030] In one embodiment, a mesenchymal stem cell culture medium further comprises raw materials in the following parts by mass: 60% of a basal medium, 25% of a serum-free medium, and 25% of a special additive. The basal medium comprises 34% of serum, 12% of fetal bovine serum, 8% of human platelet lysate, 12% of a growth factor supplement, 16% of basic fibroblast growth factor, 14% of epidermal growth factor, 5.5% of an antibiotic, 4.5% of penicillin, 7% of streptomycin, and 8% of L-glutamine; the special additive comprises 13% of an anti-differentiation agent, 14% of an antioxidant, and 18% of a directed differentiation inducer;

[0031] The anti-differentiation agent includes a ROCK inhibitor; the antioxidant includes vitamin C; the directed differentiation inducer includes an osteogenic differentiation inducer, an adipogenic differentiation inducer, a chondrogenic differentiation inducer, and other lineage differentiation inducers; the osteogenic differentiation inducer includes dexamethasone, β-glycerophosphate, ascorbic acid, and vitamin D3; the adipogenic differentiation inducer includes dexamethasone, 3-isobutyl-1-methylxanthine, insulin, and indomethacin; the chondrogenic differentiation inducer includes TGF-β3, dexamethasone, ascorbic acid, insulin-transferrin-selenium, and sodium pyruvate; the other lineage differentiation inducers include a myogenic differentiation inducer and a neuron-like differentiation inducer.

[0032] Experiment

[0033] After continuous cell culture for 7 days in Comparative Examples 1-5, a Countstar cell counter was used to count the number of adipose mesenchymal stem cells obtained by culture. The initial inoculation amount of cells in each T75 culture flask was 1.0×10 5 cells, and the results are shown in Table 1.

[0034] Table 1

[0035] Group <![CDATA[Mesenchymal stem cell number (1.0×10 5 )]]> Example 1 48.9 Example 2 35.6 Example 3 34.9 Example 4 16.8 Example 5 29.4

[0036] As can be seen from Table 1, the number of mesenchymal stem cells harvested in Example 1 is the largest, indicating that the culture medium in Example 1 has the best effect on promoting the proliferation of mesenchymal stem cells. Example 1 can significantly promote the proliferation of mesenchymal stem cells, and a large number of cells can be harvested in a short time to meet clinical needs.

[0037] Test

[0038] The test scheme is as follows:

[0039] Reference method Comparison of the amplification ability and cell phenotype differences of mesenchymal stem cells from different mouse strains.

[0040] To study the success rate of the transformation of mesenchymal stem cells into adipocytes, the results are as follows:

[0041] Example 1: 75.4%;

[0042] Example 2: 73.5%;

[0043] Example 3: 70.6%;

[0044] Example 4: 65.7%;

[0045] Example 5: 68.4%;

[0046] From the results of the above test scheme, it can be seen that the success rate of converting mesenchymal stem cells cultured in the mesenchymal stem cell medium prepared in Example 1 into adipocytes is higher, indicating that the mesenchymal stem cell medium prepared in Example 1 has better use effect and meets the clinical needs.

[0047] An application of a mesenchymal stem cell medium, wherein the mesenchymal stem cell medium is used for culturing mesenchymal stem cells.

[0048] In the present invention, the prepared mesenchymal stem cell medium can maintain its proliferation and multi-directional differentiation potential by providing appropriate nutrients and growth factors; maintain its self-renewal ability, multi-directional differentiation potential and genomic stability; by systematically optimizing the medium components and culture conditions, the yield and functional consistency of mesenchymal stem cells can be significantly improved.

[0049] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and all should be covered within the protection scope of the present invention.

Claims

1. A mesenchymal stem cell culture medium, characterized in that, It includes raw materials in the following parts by mass: 50%-70% of basal medium, 20%-30% of serum-free medium, and 10%-20% of special additive. The basal medium includes 32%-40% of serum, 10%-13% of fetal bovine serum, 7%-12% of human platelet lysate, 12%-15% of growth factor supplement, 10%-22% of basic fibroblast growth factor, 10%-18% of epidermal growth factor, 3%-10% of antibiotic, 2%-8% of penicillin, 7%-13% of streptomycin, and 6%-10% of L-glutamine; the special additive includes 10%-21% of anti-differentiation agent, 10%-22% of antioxidant, and 12%-20% of directed differentiation inducer.

2. The mesenchymal stem cell culture medium according to claim 1, wherein It also includes raw materials in the following parts by mass: 40%-70% of basal medium, 25%-30% of serum-free medium, and 10%-25% of special additive. The basal medium includes 32%-40% of serum, 11%-15% of fetal bovine serum, 6%-15% of human platelet lysate, 10%-16% of growth factor supplement, 10%-21% of basic fibroblast growth factor, 11%-20% of epidermal growth factor, 4%-8% of antibiotic, 3%-10% of penicillin, 4.5%-10% of streptomycin, and 5%-10% of L-glutamine; the special additive includes 10%-20% of anti-differentiation agent, 10%-20% of antioxidant, and 10%-20% of directed differentiation inducer.

3. The mesenchymal stem cell culture medium according to claim 1, wherein It also includes raw materials in the following parts by mass: 45%-70% of basal medium, 20%-32% of serum-free medium, and 12%-20% of special additive. The basal medium includes 30%-45% of serum, 10%-16% of fetal bovine serum, 5%-16% of human platelet lysate, 10%-18% of growth factor supplement, 8%-20% of basic fibroblast growth factor, 12%-20% of epidermal growth factor, 3%-10% of antibiotic, 2%-8% of penicillin, 5%-10% of streptomycin, and 5%-12% of L-glutamine; the special additive includes 12%-20% of anti-differentiation agent, 10%-20% of antioxidant, and 10%-23% of directed differentiation inducer.

4. A mesenchymal stem cell culture medium according to claim 1, wherein It also includes raw materials in the following parts by mass: 50%-65% of basal medium, 15%-35% of serum-free medium, and 15%-20% of special additive. The basal medium includes 32%-45% of serum, 11%-16% of fetal bovine serum, 5%-15% of human platelet lysate, 9.5%-14.5% of growth factor supplement, 10%-22% of basic fibroblast growth factor, 12%-20% of epidermal growth factor, 3.5%-8.5% of antibiotic, 4%-9% of penicillin, 5%-12% of streptomycin, and 3%-10% of L-glutamine; the special additive includes 13%-20% of anti-differentiation agent, 15%-20% of antioxidant, and 12%-20% of directed differentiation inducer.

5. A mesenchymal stem cell culture medium according to claim 1, wherein It also includes raw materials in the following parts by mass: 35%-60% of a basal medium, 25%-40% of a serum-free medium, and 20%-30% of a special additive. The basal medium includes 30%-40% of serum, 10%-15% of fetal bovine serum, 5%-15% of human platelet lysate, 10%-15% of a growth factor supplement, 10%-20% of basic fibroblast growth factor, 10%-20% of epidermal growth factor, 3%-8% of an antibiotic, 3%-8% of penicillin, 5%-10% of streptomycin, and 5%-10% of L-glutamine; the special additive includes 10%-20% of an anti-differentiation agent, 10%-20% of an antioxidant, and 10%-20% of a directed differentiation inducer.

6. The mesenchymal stem cell culture medium according to claim 1, wherein The anti-differentiation agent includes a ROCK inhibitor.

7. A mesenchymal stem cell culture medium according to claim 1, wherein The antioxidant includes vitamin C.

8. The mesenchymal stem cell culture medium according to claim 1, wherein The directed differentiation inducer includes an osteogenic differentiation inducer, an adipogenic differentiation inducer, a chondrogenic differentiation inducer, and other lineage differentiation inducers.

9. A mesenchymal stem cell culture medium according to claim 8, characterized in that, The osteogenic differentiation inducer includes dexamethasone, β-glycerophosphate, ascorbic acid, and vitamin D3; the adipogenic differentiation inducer includes dexamethasone, 3-isobutyl-1-methylxanthine, insulin, and indomethacin; the chondrogenic differentiation inducer includes TGF-β3, dexamethasone, ascorbic acid, insulin-transferrin-selenium, and sodium pyruvate; the other lineage differentiation inducers include a myogenic differentiation inducer and a neuron-like differentiation inducer.

10. Use of a mesenchymal stem cell culture medium, characterized in that, The mesenchymal stem cell medium according to any one of claims 1-9 above is applied to culturing mesenchymal stem cells.